There are many physiological and pathological conditions of animal tissue where the supply of exogenous deoxyribonucleosides may have useful therapeutic applications. In the treatment of wounds, repair of liver tissue, promotion of repair and survival after radiation, and numerous other conditions, the supply of DNA and/or deoxyribonucleosides at a high and sustained level may substantially improve the natural DNA and tissue repair processes of the affected cells.
In promoting wound healing, liver regeneration, recovery from radiation damage, and in other pathological and physiological conditions, it is likely that exogenously supplied DNA serves merely as a storage depot for deoxyribonucleosides. That depot gradually releases deoxyribonucleotides and deoxyribonucleosides during enzymatic degradation. Thus the administration of deoxyribonucleosides or derivatives disclosed herein may have value as a method for delivering those deoxyribonucleosides to tissues, which method is preferable to the administration of foreign DNA insofar as wound healing, tissue regeneration, recovery from irradiation, and the like, is concerned.
A number of investigators have attempted to use DNA and/or deoxyribonucleosides to treat a variety of conditions in experimental animals and to enhance or augment cellular repair processes, including DNA repair. It has been demonstrated that administration of exogenous DNA to experimental animals after exposure to ionizing radiation can result in dramatically increased survival and functional recovery. Studies on cell cultures in vitro demonstrate that the actual restorative agents are probably deoxyribonucleosides, the enzymatic degradation products of DNA. These compounds enhance the actual repair of damaged DNA in vitro. However, depolymerized DNA or deoxyribonucleosides administered to animals were ineffective in promoting survival or recovery after irradiation. Kanazir et al., Bull. Inst. Nuc. Sci "Boris, Kidrinch" 9:145-153 (1959). There is reason to believe that this apparent contradiction is due to the rapid catabolism of deoxyribonucleosides in vivo by the liver and other organs. Thus, after administration of deoxyribonucleosides, tissues were only exposed to effective concentrations for a matter of minutes. Beltz, et al., Bioch. Biophys. Acta297:258-267 (1973). In cell cultures, optimum survival after irradiation was found when deoxyribonucleosides were present in the incubation medium for at least 3 hours. When DNA is administered by intraperitoneal injection, it is gradually depolymerized to give a sustained release of free deoxyribonucleosides into the circulation. DNA is not, however, a suitable pharmaceutical agent to administer to humans, either orally or parenterally.
Hunting, D. J., et al., Carcinogenesis 6:1525-1528 (1985), disclose that deoxyribonucleotide synthesis is rate limiting for excision repair of UV-induced DNA damage. The authors found that there was an increase in repair ligation in cells made permeable to added deoxyribonucleotide triphosphates.
Golba, S., et al., Int. J. Rad. Biol. 13:261-268 (1967), disclose that after whole-body irradiation, administration of heterologous DNA imp,roved survival and accelerated the rate of recovery of body weight and of red blood cells, granulocytes and lymphocyte counts in the peripheral blood. No secondary disease or change in the blood count was observed in the next 12 months. Goh, K., Proc. Soc. Exp. Biol. Med. 145:938-943 (1974), discloses addition of exogenous deoxyribonucleotides resulted in prevention or healing of "pulverized" chromosomes found in cultures of leukocytes taken from a human subjected to accidental exposure to fast neutron and gamma irradiation. Horikawa, M., et al., Exp. Cell Res. 34:198-200 (1964), disclose the effect of the addition of various cell extracts and compounds to an incubation medium containing mouse L cells in culture which were irradiated in culture with X-irradiation (2000 R). Homogenates of L cells, L cell nuclei, or purified DNA from either L cells or salmon sperm all strongly enhanced the survival of the irradiated cells. RNA from either yeast or L cells was found to be ineffective. The authors suggest that the DNA hydrolysates (e.g., deoxynucleotides) are the actual reactivating agents, since heterologous DNA is as effective as homologous DNA.
Pantic, V., et al., Nature 193:993-994 (1962), disclose administration of DNA to X-irradiated rats given lethal doses of radiation. The authors found that while DNA treatment did not totally prevent cellular damage in the intestine and liver after irradiation, tissue structure and function were much closer to normal in DNA-treated animals examined 4 or 9 days after irradiation than in untreated irradiated controls.
Paoletti, C., et al., Rev. Francais. Etudes Clin. et Bio. 9:950-955 (1964), disclose a study on the effect of administration of DNA and 2-aminoethyl-isothiouronium (AET) to rats. Mice were given a mixture of AET and thiogel orally, then irradiated (700 rad) and subsequently given i.p. injections of 1 mg calf thymus DNA. The mice receiving the DNA injections recovered their weight and initial leukocyte counts more rapidly than mice similarly treated but not receiving the DNA injections.
Petrovic, D., et al., Int. J. Radiat. Biol. 18:243-258 (1970), disclose evidence concerning the molecular basis of the restorative effect of DNA in cultured mammalian cells. The authors found that the survival of irradiated cells in culture was enhanced by the addition of either DNA or equimolar amounts of deoxyribonucleosides. DNA was effective only if serum containing active deoxyribonuclease was present in the incubation medium. Thus, the authors concluded that the deoxyribonucleosides were probably the actual reactivating factors responsible for repair of radiation-induced damage. In another study, Petrovic disclosed that maximal restoration is attained when deoxyribnucleosides are in the incubation medium for at least 3 hours after irradiation. The best restoration was achieved with either a mixture of all four major deoxyribonucleosides, or a combination of deoxyguanosine with either deoxyadenosine or deoxycytidine. Petrovic, D., et al., Studia Biophysica 43:13-18 (1974). Petrovic et al. also report that in irradiated HeLa cells, treatment with a mixture of the four major deoxyribonucleosides increased survival. Petrovic et al., Int. J. Radiat. Res. 11:609-611 (1967).
Savkovic, N., Nature 203:1297-1298 (1964), discloses that 8 or 17 day old rats subjected to X-radiation (600 rem), and immediately treated with homologous testes DNA, had a much higher fertility rate than did untreated irradiated controls. Histological studies demonstrated that DNA treatment after irradiation markedly protected the structural integrity of the testes and the function of the spermatogenic processes. Savkovic also reported that heterologous DNA extracted from various organs of adult rats was effective in enhancing the survival of mice subjected to irradiation. The DNA reduced the effects of radiation by a factor of 9 to 13. Savkovic, N., et al., Nature211:1179-1180 (1966). Savkovic, N., et al., Int. J. Rad. Biol. 9:361-368 (1965) also disclose that treatment of irradiated rats with homologous DNA, isolated from liver, thymus and spleen, increased survival and fertility of the survivors. The death rate of the progeny of the irradiated rats was strongly reduced in the case of animals that received DNA after irradiation.
In another study, exposure of cultured calf liver cells to X-radiation was found to cause chromosomal damage. When cells were incubated with either DNA or equimolar concentration of deoxyribonucleotides after irradiation, there was a marked reduction in the incidence of chromosome damage. A mixture of dAMP and dGMP was as effective as a mixture of all four major deoxyribonucleotides. Ribonucleotides were ineffective in preventing radiation-induced chromosome damage. Smets, L.A., et al., Int. J. Rad. Biol. 13:269-273 (1967).
In a related study, administration of dCMP or dTMP to irradiated mice was found to improve the restoration of hematopoietic function. Soska, J., et al., Folia Biologica5:190-198 (1959).
In another study of mice irradiated with gamma radiation, administration of either a yeast RNA hydrolysate, an equimolar mixture of 3'-nucleotides or a mixture of nucleosides resulted in a significant prolongation of life span. However, long-term survival was not enhanced. The nucleic acid derivatives were administered 30 minutes, 2 days, and 4 days after irradiation. The author observed that the nucleosides, nucleotides, and RNA hydrolysate did not increase the number of surviving stem cell colonies in spleen or bone marrow, but rather appeared to improve the functional capacity of irradiated cells during the critical period after irradiation. These compounds also appeared to accelerate the process of maturation and differentiation of the progeny of surviving stem cells. Sugahara, T., et al., Brookhaven Symposia in Biology, 284-302 (1967).
In a study of guinea pigs subjected to X-radiation, animals given RNA or ATP immediately before and after irradiation had much higher 21-day survival rates than did untreated irradiated controls. Most of the animals that survived the 21-day observation period recovered fully, with no secondary radiation-induced disease. Wagner, R., Int. J. Rad. Biol. 2:101-112 (1967).
In another study, administration of DNA from different sources, including calf thymus, rat liver and spleen, herring and salmon sperm, and Ehrlich ascites carcinoma cells was studied in rats given lethal doses of gamma irradiation. All forms of DNA significantly increased the survival of the irradiated rats. The quantitative differences in the effects of the DNA from different sources were directly related to the molecular weight. The authors found a reduction in therapeutic efficiency which is proportional to the reduction in molecular size upon DNA shearing. Wilczok, T., et al., Int. J. Rad. Biol. 9:201-211 (1965).
The incidence of chromosomal abnormalities in lymphocytes from radiologists chronically exposed to X-rays, was determined before, during, and after treatment with DNA and ATP. The basal incidence of chromosomal damage was substantially higher than in unexposed control subjects. Daily injection of DNA and ATP resulted in 2 to 3 fold decreases in the frequency of chromosomal abnormalities. Following discontinuation of treatment, the incidence of chromosomal damage returned toward pretreatment levels. (Goyanes-Villaescusa, Lancet II:575 (1973).
There have also been reports on the use of DNA preparations to treat wounds. For example, Dumont, Ann. Surg. 150:799 (1959), disclose that exogenous DNA, applied to experimental wounds in rabbit ears, accelerated the growth of granulation tissue in the wounds. A mixture of DNA plus deoxyribonuclease (the enzyme primarily responsible for degradation of DNA) was more effective in accelerating fibroplasia than either DNA or deoxyribonuclease alone. The total amount of granulation tissue formed after treatment with DNA was not greater than in untreated controls; the onset and rate of its growth were however significantly accelerated. The authors suggest that low polymer DNA fragments are the actual active agents.
Nicolau et al., Der Hautartzt 17:512 (1966), disclose a study on experimental skin wounds on the backs of rats which were treated daily with a 1% solution of DNA in physiological saline. The wounds treated with local application of DNA were cicatrized within four to eight days; those treated only with physiological saline were cicatrized only after 10 to 15 days.
Marshak et al., Proc. Soc. Exp. Biol. Med. 58:62 (1945), disclose that application of DNA to experimental skin wounds in rats resulted in a significant acceleration of the growth of granulation tissue within the wounds, as compared to untreated controls. Although the granulation tissue appeared sooner in treated wounds, the final amount of granulation tissue was not abnormal.
Newman et al., Am. J. Physiol. 164:251 (1951), disclose a study of rats subjected to partial hepatectomy. The course of liver regeneration was followed for 11 days. The livers of rats treated with DNA regenerated significantly faster than livers in untreated animals. RNA treatment also accelerated liver regeneration, though not as markedly as DNA administration.
Certain derivatives of deoxyribonucleosides have been prepared. Casida et al., Biochemical Pharmacology vol. 15, p. 627-644, 1966, describe the preparation of the 3'5'-diacetyl, dipropionyl and dibutyryl esters of 2'-deoxythymidine. Rosowsky et al., Cancer Treatment Reports vol. 65 No.1-2, p. 93-99, January/February 1981, and Ensminger et al., Biochemical Pharmacology vol. 28, p. 1541-1545, October 1978, describe the use of thymidine 5'-O-pivaloate to supply thymidine to tissues.
Since the primary determinant of recovery or survival after exposure to ionizing radiation or chemical mutagens is the preservation or repair of DNA, a number of compounds have been found which, when present in an organism at the time of exposure to radiation or chemical mutagens, attenuate the damage to DNA and other cellular structures. Included in this class of compounds are antioxidants, sulfhydryl compounds, and the enzymes superoxide dismutase and catalase. However, these compounds have been found to be only moderately protective or practical to use in vivo, in part because they can be toxic in effective concentrations. Since these compounds must be present in the organism at the time of exposure to radiation or chemical mutagens, they are obviously not useful in the case of unexpected or accidental exposure.
Reportedly, sulfhydryl compounds are the most effective radioprotective agents known. Examples of these compounds include mercaptoethylamine (MEA), 2-.beta.-amino-ethyl-isothiouronium-Br-HBr (AET), 5-hydroxytryptamine (HT), and 5-2-(3-amino-propylamino)ethylphosphorothiotoic acid (WR-2721). However, many of these compounds are toxic. Thus, several investigators have attempted to increase. protection against radiation damage and to decrease-toxicity by using mixtures of these chemical protectors. The results of these studies demonstrate that the administration of mixtures of radioprotectors not only increases the degree of protection for short and long term survival compared with that from each substance given separately, but also diminishes the toxicity of compounds such as AET or MEA. Administration of sulfhydryl chemical radioprotectors before exposure to radiation diminishes markedly the changes induced by radiation in the structures. Maisin, J. R., in: Symposium on Perspectives in Radioprotection, Armed Forces Radiobiology Research Institute, Bethesda, Maryland, p. 53 (1987).
Thiols reportedly protect DNA by mechanisms comprising hydroxyl radical scavenging and DNA radical repair mechanisms. Thus, the extent of interactions of thiols with DNA determines the amount of protection. Cationic thiols (2-[(aminopropyl)amino]ethanethiol (WR-1065) and cysteamine) are better protectors than neutral thiol (2-mercaptoethanol and dithiothreitol) which are in turn better protectors than anionic thiols (glutathione (GSH), 2-mercaptoethanesulfonic acid, and mercaptosuccinate). Such differential binding provides a basis for understanding why WR-1065, which scavenges hydroxyl radicals at a rate comparable to that for GSH, effectively protects cells at concentrations well below those of GSH. Fahery, R. C., ibid., p. 31.
In studies of Chinese hamster V-79 cells treated with gamma radiation and with bromodeoxyuridine (BrdUrd) and light photolysis were compared. When treated with gamma radiation, WR-2721 was found to improve cell survival both by acting as a reducer of gamma radiation, and by causing increase in DNA repair and increase in rejoining of DNA strand breaks. Cysteamine has been shown to act as a reducer of gamma radiation damage without affecting the rejoining of strand breaks or DNA repair capacity. Nicotinamide (NA) has been shown to directly affect DNA repair through the polyADPribose system which is activated by DNA single strand breaks, thus providing NA concentration dependent protection or sensitization. These compounds exhibit a different effect on cells treated with BrdUrd and light compared with gamma radiation. WR-2721 does not reduce strand break formation. MEA and NA reduce damage formation by about 30%. WR-2721 did not affect the rejoining of BrdUrd/light-induced DNA strand breaks. Only NA increased the repair capability of cells subjected to BrdUrd and light damage. Prager, A., et al., ibid., p. 43.
In addition to the use of thiols, radioprotection has been achieved with "biological response modifiers" (BRM), either alone or in combination with other agents. Such biological response modifiers include glucan, OK-432, Biostim, PSK, Lentinan, Schizophyllan, Rhodexman, Levan, Mannozym, and MVE-2. Of these BRM's, glucan was found to be the most radioprotective. Glucan is a beta 1-3 polyglycan isolated from the yeast Saccharomyces cerevisiae. Glucan's radioprotective capacity is attributed to its ability both to protect and/or enhance recovery of hemoatopoietic stem cell populations, and to enhance or maintain the function of macrophage cell populations important in combatting otherwise lethal post-irradiation opportunistic infections. The combination of glucan and WR-2721 resulted in both additive and synergistic radioprotective effects. Patchen, M. L., ibid., p. 68.
Other polysaccharides have also been found to be radioprotective. Intravenous administration of the polysaccharide extracted from the yeast Rhodotorula rubra, mannane mannozyme (MMZ), and the particulate polyglucans GLP/B04 and GLP/B05 (unbranched glucans with alternating B-1,3 and B-1,6 bonds), significantly decreased the mortality of mice exposed to a single dose of X-rays. Maisin, J. R., ibid., p. 69.
The cytokines IL-2 and TNF have also been found to be effective radioprotective compounds. Cytokines are released upon administration of numerous inflammatory agents. Many of these inflammatory agents stimulate the reticuloendothelial system and are radioprotective. Neta, R., ibid, p. 71.
Thymic peptides, such as thymic factor TF-5, have also been reported to reverse or greatly ameliorate immune depression due to limited portal irradiation of thymus, circulating blood, and lymphoid tissues. The immune restorative effect of thymic factors is due to their maturational effect on bone marrow immunocyte precursors. Chretien, P. B., ibid., p. 72.
The antioxidant enzymes glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase, glutathione reductase and glutathione transferase scavenge free radical species produced by radiation and/or the products of free radical cellular damage, and thus play a role in radioprotection. GSH-Px exhibits and best correlation between enzyme activity and cell radiosensitivity. Administration of enzyme preparations or drugs or chemicals which mimic or activate or induce these enzymes may enhance radioprotection. The radioprotectors MEA, WR-2721 and diethyldithiocarbamate (DDC) enhance mouse liver GSH-Px activity 1 to 2 hours after administration. Selenium and selenium-containing compounds also exhibit a small radioprotective effect. The levels of GSH-Px in mouse bone marrow were found to increase 30% 24 hours after administration of selenium. When selenium was administered before WR-2721, a decrease in toxicity and an increase in radioprotection was observed. Superoxide dismutase (SOD) and catalase were also observed to increase upon administration of selenium. In addition, metal ions and metal-containing compounds which mimic antioxidant enzymes may also act as radioprotectors. Copper and zinc metal ions in SOD are marginally radioprotective. Mimetics of SOD include bis (3,5-diisopropylsalicylato) copper and the bivalent copper complex of 3-mercapto-2-hydroxypropylether of dextran. Dumar, K. S., ibid., p. 89. For a review on SOD, see Fridovich, I., Annu. Rev. Biochem. 44: 147-159 (1975).
Induction of metallothionein (MT) in the body by treatment with some heavy metals or immunostimulants has been found to be a potent means for inducing radioprotection. The metal salts CdCl.sub.2, MnCl.sub.2 or zinc acetate or the immunostimulants OK-432 or IL-1 elevates MT levels in the liver of pretreated mice 10 to 20 times of the control level. The number of leukocytes as well as erythrocytes were reduced temporarily even in pretreated mice. However, the cell counts of pretreated mice showed a faster recovery. Matsubara, J., et al., ibid., p. 99.
Vitamin A and beta carotene have also been suggested as radioprotective agents. They may be involved in ameliorating the oxidative damage in tissues of irradiated mammals which results from production of free radicals such as hydroxy radicals or H.sub.2 O.sup.+ and its daughter products. Radiation may also create injury to cell structures either by the direct effect of radiation or by the production of toxic metabolites. Radiation injury results in disturbed extracellular and intracellular oxygen levels and perturbed intracellular electron transport and metabolism. Oxidative damage may be enhanced by sudden elevations of local oxygen levels caused by reperfusion of tissues after radiation-induced vasoconstriction or by reversal of radiation-induced bronchoconstriction. Damage occurs where local oxygen levels are in excess of what the tissues can consume. Vitamin A and beta carotene were found to exhibit protective action in rodents exposed to whole body and local radiation. Seifter, E., et al., ibid. p. 104.
Prostaglandins and related compounds of the arachidonic acid cascade protect cells in vivo from some degree of ionizing radiation injury. Among the array of physiological actions of prostaglandins is the protection of cells and tissues from a variety of injuries including strong acids, bases, and absolute ethanol. Prostaglandins were found to exhibit maximal protection at levels a thousandfold lower than those needed for WR-2721. Hanson, W. R., ibid., p. 105.
Methylene blue, a compound used clinically as an anti-inflammatory, antimalarial, and antibacterial agent as well as in the treatment of carbon monoxide poisoning and as an antidote for cyanide poisoning, as also found to protect the intestinal mucosa of rats subjected to sublethal radiation-induced damage. Irradiation damages tissues through the production of highly bioactive free radical species. Therefore, it was hypothesized that methylene blue would also protect irradiated rats from free-radical mediated tissue damage. Scheving, L. E., et al., ibid., p. 115.
In addition, N-arylacetyldehydroalanines reportedly inhibit superoxide anion and hydroxyl free radical-mediated processes, thereby providing radioprotective activity. Buc-Calderone, P., et al., ibid., p. 116.